Nerve growth factor (NGF) is a protein that affects the development and maintenance of sympathetic and certain sensory neurons. We propose to use cell cultures of sympathetic neurons and of the PC12 and other clonal lines of pheochromocytoma cells to study a) the possible role and types of action of NGF in early sympathicoblast/sympathetic neuron development, b) the consequences of NGF's actions on its target cells, and above all, c) the primary and secondary mechanism(s) of action whereby NGF promotes its actions. With regard to mechanism, the types of actions to be studied include effects on neuronal survival, initiation and maintenance of neurite outgrowth and elongation, promotion of neurite regeneration, and regulation of enzyme activity. Specific projects include effects of NGF on cell surface architecture, the importance of formation and internalization of NGF/NGF-receptor complexes; a pilot project on feasibility of purifying NGF receptors from the PC12 cell line; testing the hypothesis that NGF promotes survival via regulation of metabolite uptake; simplification of the PC12 cell NGF bioassay; documenting and studying the mechanism of action of the regulation of acetylcholinesterase activity by NGF; NGF's effect on the synthesis and/or chemical properties of specific cell proteins and the relation of such proteins to NGF's mechanism of action; effects of NGF on the assembly and/or organization of microtubules; the use of pheochromocytoma variants and mutants to genetically dissect NGF's mechanisms of action; stimulation of cell proliferation by NGF; and maintenance, expansion and distribution of our stocks of PC12 cells.